Polynucleotides and polypeptides involved in post-transcriptional gene silencing

ABSTRACT

This invention relates to isolated nucleic acid fragments encoding polypeptides involved in post-transcriptional gene silencing. The invention also relates to construction of a recombinant DNA construct encoding all or a portion of the polypeptide involved in post-transcriptional gene silencing, in sense or antisense orientation, wherein expression of the recombinant DNA construct results in production of altered levels in a transformed host cell of the the polypeptide involved in post-transcriptional gene silencing.

This application claims the benefit of U.S. Provisional Application No.60/298,973, filed Jun. 18, 2001, the entire content of which is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingpolypeptides in plants and seeds involved in post-transcriptional genesilencing.

BACKGROUND OF THE INVENTION

Post-transcriptional gene silencing (PTGS), which operates at the levelof sequence-specific RNA degradation, has emerged as a major phenomenonthrough which transgene expression in plants is down-regulated. It wasfirst recognized in plants, and similar mechanisms since then have beenobserved in non-plant systems, where it is known by different names, towit, quelling in the fungus Neurospora crassa (Romano and Macino (1992)Mol Microbiol 6:3343-3353), and RNA interference (RNAi) in worms, flies,and mammals (Bosher and Labouesse (2000) Nat Cell Biol 2:E31-36).

Although the mechanism remains to be fully elucidated, it appears thatdouble-stranded RNA (dsRNA) serve as key intermediates in PTGS (Bass(2000) Cell 101:235-238). The involvement of dsRNA is supported byidentification of small complementary RNA (cRNA), 21-25 nucleotideslong, which can bind the target RNA to form dsRNA, in PTG-silencedplants (Hamilton and Baulcombe (1999) Science 286:950-952), and thefinding that a protein similar to RNA-dependent RNA polymerase, theenzyme involved in cRNA synthesis, is required for PTGS (Mourrain et al.(2000) Cell 101:533-542).

Another protein identified to be required for PTGS is the ARGONAUTE(AGO1) protein (Bohmert et al. (1998) EMBO J. 17:170-180; Fagard et al.(2000) Proc Natl Acad Sci USA 97:11650-11654). AGO1 protein shareshomology with the RDE1 and QDE-2 proteins which have been found to berequired for RNAi in C. elegans and for quelling in Neurospora,respectively, thus reinforcing the notion that PTGS, RNAi, and quellingare similar processes at the mechanistic level. AGO1/RDE1/QDE-2 proteinsare similar to eIF2C, a protein important for protein translation. It istherefore hypothesized that dsRNA mediates PTGS by disrupting properpositioning of eIF2C in the translation machinery complex, therebypreventing translation of the target mRNA (Tabara et al. (1999) Cell99:123-132; Fagard et al. (2000) Proc Natl Acad Sci USA 97:11650-11654).

It is apparent that PTGS is an important process, which if manipulatedproperly, may be used to control transgene expression. Disclosed hereinare sequences very homologous to the AGO1 protein family, which includesthe ZWILLE (ZLL) or PINHEAD (PNH) protein involved in plant development(Moussian et al. (1998) EMBO J 17:1799-1809; Lynn et al. (1999)Development 126:469-481), and the RDE-1 protein involved in transposonsilencing (Tabara et al. (1999) Cell 99:123-132). These sequences may beused to manipulate PTGS. Since some of the AGO1 family members have alsobeen shown to be involved in transposon silencing, meristem development,and differentiation of meristematic tissue, the polynucleotidesdisclosed herein may also be used to manipulate transposon activity,meristem activity, plant architecture and development, and proliferationof undifferentiated plant cells in culture, which would be useful incallus propagation.

SUMMARY OF INVENTION

The present invention includes isolated polynucleotides comprising: (a)a first nucleotide sequence encoding a first polypeptide havingpost-transcriptional gene silencing activity wherein the amino acidsequence of the first polypeptide and the amino acid sequence of SEQ IDNO:12, 14, 22, 28, 40 or 54 have at least 80% sequence identity, or (b)a second nucleotide sequence encoding a second polypeptide havingpost-transcriptional gene silencing activity wherein the amino acidsequence of the second polypeptide and the amino acid sequence of SEQ IDNO:8, 38 or 42 have at least 85% sequence identity. For the firstpolypeptide, it is preferred that the identity be at least 85%, it ismore preferred that the identity is at least 90%, and it is even morepreferred that the identity be at least 95%. For the second polypeptide,it is preferred that the identity be at least 90%, and it is morepreferred that the identity be at least 95%. More preferably, thepresent invention includes isolated polynucleotides encoding the aminoacid sequence of SEQ ID NO:8, 12, 14, 22, 28, 38, 40, 42 or 54 ornucleotide sequences comprising the nucleotide sequence of SEQ ID NO:7,11, 13, 21, 27, 37, 39, 41 or 53. The present invention also includesisolated polynucleotides comprising the complement of nucleotidesequences of the present invention.

The present invention also includes:

-   -   in a preferred first embodiment, an isolated polynucleotide        comprising: (a) a first nucleotide sequence encoding a first        polypeptide, wherein the amino acid sequence of the first        polypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22,        28, 40 or 54 have at least 80%, 85%, 90%, or 95% sequence        identity, (b) a second nucleotide sequence encoding a second        polypeptide, wherein the amino acid sequence of the second        polypeptide and the amino acid sequence of SEQ ID SEQ ID NO:8,        38 or 42 have at least 85%, 90%, or 95% sequence identity,        or (c) the complement of the nucleotide sequence of (a) or (b);        the first polypeptide preferably comprises the amino acid        sequence of of SEQ ID NO:12, 14, 22, 28, 40 or 54; the second        polypeptide preferably comprises the amino acid sequence of SEQ        ID NO:8, 38 or 42; the first nucleotide sequence preferably        comprises the nucleotide sequence of SEQ ID NO:11, 13, 21, 27,        39 or 53; the second nucleotide sequence preferably comprises        the nucleotide sequence of SEQ ID NO:7, 37 or 41; the first and        second polypeptides preferably have post-transcriptional gene        silencing activity;    -   in a preferred second embodiment, a recombinant DNA construct        comprising any of the isolated polynucleotides of the present        invention operably linked to at least one regulatory sequence,        and a cell, a plant, and a seed comprising the recombinant DNA        construct;    -   in a preferred third embodiment, a vector comprising any of the        isolated polynucleotides of the present invention;    -   in a preferred fourth embodiment, an isolated polynucleotide        comprising a nucleotide sequence comprised by any of the        polynucleotides of the first embodiment, wherein the nucleotide        sequence contains at least 30, 40, or 60 nucleotides;    -   in a preferred fifth embodiment, a method for transforming a        cell comprising transforming a cell with any of the isolated        polynucleotides of the present invention, and the cell        transformed by this method, advantageously, the cell is        eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a        bacterium;    -   in a preferred sixth embodiment, a method for producing a        transgenic plant comprising transforming a plant cell with any        of the isolated polynucleotides of the present invention and        regenerating a plant from the transformed plant cell, a        transgenic plant produced by this method, and seed obtained from        this transgenic plant;    -   in a preferred seventh embodiment, an isolated polypeptide        comprising: (a) a first amino acid sequence, wherein the first        amino acid sequence and and the amino acid sequence of SEQ ID        NO:12, 14, 22, 28, 40 or 54 have at least 80%, 85%, 90% or 95%        sequence identity, or (b) a second amino acid sequence, wherein        the second amino acid sequence and and the amino acid sequence        of SEQ ID NO:8, 38 or 42 have at least 85%, 90% or 95% sequence        identity; the first amino acid sequence preferably comprises the        amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54, and        the second amino acid sequence preferably comprises the amino        acid sequence of SEQ ID NO:8, 38 or 42; the polypeptide        preferably has post-transcriptional gene silencing activity;    -   in a preferred eight embodiment, a method for isolating a        polypeptide encoded by polynucleotides of the present invention        comprising isolating the polypeptide from cultivated cells, from        the culture medium, or from both the cultivated cells and the        culture medium, wherein the cells contain a recombinant DNA        construct comprising the polynucleotide operably linked to at        least one regulatory sequence;    -   in a preferred ninth embodiment, a virus, preferably a        baculovirus, comprising any of the isolated polynucleotides of        the present invention or any of the recombinant DNA constructs        of the present invention;    -   in a preferred tenth embodiment, a method of selecting an        isolated polynucleotide that affects the level of expression in        a host cell, preferably a plant cell, of a gene encoding a        polypeptide having post-transcriptional gene silencing activity,        the method comprising the steps of: (a) constructing an isolated        polynucleotide of the present invention or an isolated        recombinant DNA construct of the present invention; (b)        introducing the isolated polynucleotide or the isolated        recombinant DNA construct into a host cell; (c) measuring the        level of the polypeptide involved in post-transcriptional gene        silencing or its activity in the host cell containing the        isolated polynucleotide or the isolated recombinant DNA        construct; and (d) comparing the level of the polypeptide        involved in post-transcriptional gene silencing or its activity        in the host cell containing the isolated polynucleotide or the        isolated recombinant DNA construct with the level of the        polypeptide involved in post-transcriptional gene silencing or        its activity in the host cell that does not contain the isolated        polynucleotide or the isolated recombinant DNA construct;    -   in a preferred eleventh embodiment, a method of obtaining a        nucleic acid fragment encoding a substantial portion of a        polypeptide involved in post-transcriptional gene silencing        comprising the steps of: synthesizing an oligonucleotide primer        comprising a nucleotide sequence of at least 30 (preferably at        least 40, most preferably at least 60) contiguous nucleotides        derived from a nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9,        11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,        43, 45, 47, 49, 51 or 53, or the complement of such nucleotide        sequences; and amplifying a nucleic acid fragment (preferably a        cDNA inserted in a cloning vector) using the oligonucleotide        primer; the amplified nucleic acid fragment preferably will        encode a substantial portion of a polypeptide involved in        post-transcriptional gene silencing;    -   in a preferred twelfth embodiment, a method of obtaining a        nucleic acid fragment encoding all or a substantial portion of        the amino acid sequence encoding a polypeptide involved in        post-transcriptional gene silencing comprising the steps of:        probing a cDNA or genomic library with an isolated        polynucleotide of the present invention; identifying a DNA clone        that hybridizes with an isolated polynucleotide of the present        invention; isolating the identified DNA clone; and sequencing        the cDNA or genomic fragment that comprises the isolated DNA        clone;    -   in a preferred thirteenth embodiment, a method for positive        selection of a transformed cell comprising: (a) transforming a        host cell with a recombinant DNA construct of the present        invention or an expression cassette of the present invention;        and (b) growing the transformed host cell, preferably a plant        cell, such as a monocot or a dicot, under conditions which allow        expression of the polypeptide involved in post-transcriptional        gene silencing polynucleotide in an amount sufficient to        complement a null mutant to provide a positive selection means;        and    -   in a preferred fourteenth embodiment, a method of altering the        level of expression of a polypeptide involved in        post-transcriptional gene silencing in a host cell        comprising: (a) transforming a host cell with a recombinant DNA        construct of the present invention; and (b) growing the        transformed host cell under conditions that are suitable for        expression of the recombinant DNA construct wherein expression        of the recombinant DNA construct results in production of        altered levels of the polypeptide involved in        post-transcriptional gene silencing in the transformed host        cell.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A, 1B, 1C and 1D depict the amino acid sequence alignment of thepolypeptides involved in post-transcriptional gene silencing encoded bythe following: (a) nucleotide sequence derived from corn clonecle1f.pk002.k13 (SEQ ID NO:8), (b) nucleotide sequence derived from cornclone p0119.cmtmm21r (SEQ ID NO:22), (c) nucleotide sequence derivedfrom soybean clone ssl1c.pk003.g3 (SEQ ID NO:40), (d) nucleotidesequence of a contig assembled from nucleotide sequences obtained fromwheat clone wdk1c.pk012.i2 and PCR fragments (SEQ ID NO:42), and (e)nucleotide sequence from Otyza sativa (NCBI GenBank Identifier (GI) No.6539559; SEQ ID NO:55). Amino acids which are conserved among all and atleast two sequences with an amino acid at that position are indicatedwith an asterisk (*). Dashes are used by the program to maximizealignment of the sequences.

FIGS. 2A, 2B, 2C, 2D and 2E depict the amino acid sequence alignment ofthe polypeptides involved in post-transcriptional gene silencing encodedby the following: (a) nucleotide sequence derived from corn clonecsc1c.pk006.j19 (SEQ ID NO:12), (b) nucleotide sequence derived fromcorn clone ctn1c.pk003.i20 (SEQ ID NO:14), (c) nucleotide sequence of acontig assembled from nucleotide sequences obtained from rice clonerlm1n.pk001.m11 and PCR fragments (SEQ ID NO:28), (d) nucleotidesequence of a contig assembled from nucleotide sequences obtained fromsoybean clone sdc2c.pk001.p4 and PCR fragments (SEQ ID NO: 38), and (e)nucleotide sequence from Arabidopsis thaliana (NCBI GenBank Identifier(GI) No. 2149640; SEQ ID NO:56). Amino acids which are conserved amongall and at least two sequences with an amino acid at that position areindicated with an asterisk (*). Dashes are used by the program tomaximize alignment of the sequences.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more EST, FIS or PCR sequences (“Contig”), or sequences encoding theentire protein, or functionally active polypeptide, derived from an EST,an FIS, or a contig (“CGS”). The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Polypeptides Involved inPost-Transcriptional Gene Silencing Polypeptide SEQ ID NO: (PlantSource) Clone Designation Status (Nucleotide) (Amino Acid) ZwilleHomolog (Corn) p0102.cerba57r FIS 1 2 Zwille Homolog (Soybean)ses2w.pk0009.g6 FIS 3 4 Zwille Homolog (Soybean) ssm.pk0063.a4 FIS 5 6Argonaute Homolog (Corn) cle1f.pk002.k13 (FIS) CGS 7 8 Argonaute Homolog(Corn) cpf1c.pk008.j24 FIS 9 10 Argonaute Homolog (Corn) csc1c.pk006.j19(FIS) CGS 11 12 Argonaute Homolog (Corn) ctn1c.pk003.i20 (FIS) CGS 13 14Argonaute Homolog (Corn) Contig of contig 15 16 p0002.cgevj06rp0125.czaab55r (FIS) p0125.czaat57r Argonaute Homolog (Corn)p0102.cerae32ra EST 17 18 Argonaute Homolog (Corn) p0107.cbcbd69r EST 1920 Argonaute Homolog (Corn) p0119.cmtmm21r (FIS) CGS 21 22 ArgonauteHomolog (Rice) rca1n.pk018.b3 FIS 23 24 Argonaute Homolog (Rice)rl0n.pk124.g8 FIS 25 26 Argonaute Homolog (Rice) Contig of CGS 27 28rlm1n.pk001.m11 (FIS) PCR fragment sequence Argonaute Homolog (Rice)rls6.pk0082.d4 FIS 29 30 Argonaute Homolog (Rice) rsl1n.pk004.d12 FIS 3132 Argonaute Homolog (Rice) rtc1c.pk008.k19.f EST 33 34 ArgonauteHomolog (Soybean) sdc1c.pk0004.d11 FIS 35 36 Argonaute Homolog (Soybean)Contig of CGS 37 38 sdc2c.pk001.p4 (FIS) PCR fragment sequence ArgonauteHomolog (Soybean) ssl1c.pk003.g3 (FIS) CGS 39 40 Argonaute Homolog(Wheat) Contig of CGS 41 42 wdk1c.pk012.i2 (FIS) PCR fragment sequenceArgonaute Homolog (Wheat) wlm96.pk029.c23 FIS 43 44 Argonaute Homolog(Wheat) wne1g.pk003.f8 EST 45 46 Argonaute Homolog (Wheat) wr1.pk0073.c7EST 47 48 Argonaute Homolog (Wheat) wre1n.pk0001.h6 FIS 49 50 ArgonauteHomolog (Wheat) wre1n.pk162.h10 EST 51 52 Argonaute Homolog (Rice)rdi2c.pk002.d14:fis CGS 53 54

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

The problem to be solved, therefore, was to identify polynucleotidesthat encode polypeptides involved in post-transcriptional genesilencing. These polynucleotides may be used in plant cells to alter thepost-transcriptional gene silencing pathway. More specifically, thepolynucleotides of the instant invention may be used to createtransgenic plants where the levels of polypeptides involved inpost-transcriptional gene silencing are altered with respect tonon-transgenic plants which would result in plants with an enhancementor a deficiency in post-transcriptional gene silencing. The presentinvention has solved this problem by providing polynucleotide anddeduced polypeptide sequences corresponding to novel polypeptidesinvolved in post-transcriptional gene silencing from corn (Zea mays),rice (Oryza sativa), soybean (Glycine max) and wheat (Triticumaestivum).

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 or 53, or the complement of suchsequences.

The term “isolated” refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques. A “recombinantDNA construct” comprises any of the isolated polynucleotides of thepresent invention operably linked to at least one regulatory sequence.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53, and thecomplement of such nucleotide sequences may be used to affect theexpression and/or function of a polypeptide involved inpost-transcriptional gene silencing in a host cell. A method of using anisolated polynucleotide to affect the level of expression of apolypeptide in a host cell (eukaryotic, such as plant or yeast,prokaryotic such as bacterial) may comprise the steps of: constructingan isolated polynucleotide of the present invention or an isolatedrecombinant DNA construct of the present invention; introducing theisolated polynucleotide or the isolated recombinant DNA construct into ahost cell; measuring the level of a polypeptide or enzyme activity inthe host cell containing the isolated polynucleotide; and comparing thelevel of a polypeptide or enzyme activity in the host cell containingthe isolated polynucleotide with the level of a polypeptide or enzymeactivity in a host cell that does not contain the isolatedpolynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least 70% identical,preferably at least 80% identical to the amino acid sequences reportedherein. Preferred nucleic acid fragments encode amino acid sequencesthat are at least 85% identical to the amino acid sequences reportedherein. More preferred nucleic acid fragments encode amino acidsequences that are at least 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are at least 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above identities but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying related polypeptidesequences. Useful examples of percent identities are 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to100%. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the ClustaIV method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the ClustaIV method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST algorithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, recombinant DNA constructs, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase 1. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632). A “mitochondrial signal peptide” is an amino acidsequence which directs a precursor protein into the mitochondria (Zhangand Glaser (2002) Trends Plant Sci 7:14-21).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277; Ishida Y. et al. (1996)Nature Biotech. 14:745-750) and particle-accelerated or “gene gun”transformation technology (Klein et al. (1987) Nature (London)327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).Thus, isolated polynucleotides of the present invention can beincorporated into recombinant constructs, typically DNA constructs,capable of introduction into and replication in a host cell. Such aconstruct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1989;and Flevin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990. Typically, plant expression vectors include, forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The term“transformation” as used herein refers to both stable transformation andtransient transformation.

The terms “recombinant construct”, “expression construct” and“recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell using standard methodology well knownto one skilled in the art. Such construct may be used by itself or maybe used in conjunction with a vector. If a vector is used, the choice ofvector is dependent upon the method that will be used to transform hostplants as is well known to those skilled in the art.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“Motifs” or “subsequences” refer to short regions of conserved sequencesof nucleic acids or amino acids that comprise part of a longer sequence.For example, it is expected that such conserved subsequences would beimportant for function, and could be used to identify new homologues inplants. It is expected that some or all of the elements may be found ina homologue. Also, it is expected that one or two of the conserved aminoacids in any given motif may differ in a true homologue.

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention includes an isolated polynucleotide comprising:(a) a first nucleotide sequence encoding a first polypeptide comprisingat least 100 amino acids, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, orSEQ ID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based onthe ClustaIV alignment method, (b) a second nucleotide sequence encodinga second polypeptide comprising at least 200 amino acids, wherein theamino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or 95%identity based on the ClustaIV alignment method, (c) a third nucleotidesequence encoding a third polypeptide comprising at least 100 aminoacids, wherein the amino acid sequence of the third polypeptide and theamino acid sequence of SEQ ID NO:34 have at least 80%, 85%, 90%, or 95%identity based on the ClustaIV alignment method, (d) a fourth nucleotidesequence encoding a fourth polypeptide comprising at least 150 aminoacids, wherein the amino acid sequence of the fourth polypeptide and theamino acid sequence of SEQ ID NO:10 have at least 80%, 85%, 90%, or 95%identity based on the ClustaIV alignment method, (e) a fifth nucleotidesequence encoding a fifth polypeptide comprising at least 200 aminoacids, wherein the amino acid sequence of the fifth polypeptide and theamino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ IDNO:50 have at least 80%, 85%, 90%, or 95% identity based on the ClustaIValignment method, (f) a sixth nucleotide sequence encoding a sixthpolypeptide comprising at least 300 amino acids, wherein the amino acidsequence of the sixth polypeptide and the amino acid sequence of SEQ IDNO:28, SEQ ID NO:40 or SEQ ID NO:54 have at least 80%, 85%, 90%, or 95%identity based on the ClustaIV alignment method, (g) a seventhnucleotide sequence encoding a seventh polypeptide comprising at least100 amino acids, wherein the amino acid sequence of the seventhpolypeptide and the amino acid sequence of SEQ ID NO:26 have at least85%, 90%, or 95% identity based on the ClustaIV alignment method, (h) aneighth nucleotide sequence encoding an eighth polypeptide comprising atleast 200 amino acids, wherein the amino acid sequence of the eighthpolypeptide and the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32have at least 85%, 90%, or 95% identity based on the ClustaIV alignmentmethod, (i) a ninth nucleotide sequence encoding a ninth polypeptidecomprising at least 250 amino acids, wherein the amino acid sequence ofthe ninth polypeptide and the amino acid sequence of SEQ ID NO:8 or SEQID NO:12 have at least 85%, 90%, or 95% identity based on the ClustaIValignment method, 0) a tenth nucleotide sequence encoding a tenthpolypeptide comprising at least 300 amino acids, wherein the amino acidsequence of the tenth polypeptide and the amino acid sequence of SEQ IDNO:42 have at least 85%, 90%, or 95% identity based on the ClustaIValignment method, (k) an eleventh nucleotide sequence encoding aneleventh polypeptide comprising at least 100 amino acids, wherein theamino acid sequence of the eleventh polypeptide and the amino acidsequence of SEQ ID NO:46 have at least 90% or 95% identity based on theClustaIV alignment method, (I) a twelfth nucleotide sequence encoding atwelfth polypeptide comprising at least 150 amino acids, wherein theamino acid sequence of the twelfth polypeptide and the amino acidsequence of SEQ ID NO:4 have at least 90% or 95% identity based on theClustaIV alignment method, (m) a thirteenth nucleotide sequence encodinga thirteenth polypeptide comprising at least 250 amino acids, whereinthe amino acid sequence of the thirteenth polypeptide and the amino acidsequence of SEQ ID NO:38 have at least 90% or 95% identity based on theClustaIV alignment method, or (n) the complement of the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, or thirteenth nucleotide sequence, wherein the complement andthe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, or thirteenth nucleotide sequence contain thesame number of nucleotides and are 100% complementary. The firstpolypeptide preferably comprises the amino acid sequence of SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ IDNO:44, SEQ ID NO:48, or SEQ ID NO:52, the second polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:24, the third polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:34, the fourthpolypeptide preferably comprises the amino acid sequence of SEQ IDNO:10, the fifth polypeptide preferably comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:22, or SEQ ID NO:50, thesixth polypeptide preferably comprises the amino acid sequence of SEQ IDNO:28, SEQ ID NO:40 or SEQ ID NO:54, the seventh polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:26, the eighthpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:14or SEQ ID NO:32, the ninth polypeptide preferably comprises the aminoacid sequence of SEQ ID NO:8 or SEQ ID NO:12, the tenth polypeptidepreferably comprises the amino acid sequence of SEQ ID NO:42, theeleventh polypeptide preferably comprises the amino acid sequence of SEQID NO:46, the twelfth polypeptide preferably comprises the amino acidsequence of SEQ ID NO:4, and the thirteenth polypeptide preferablycomprises the amino acid sequence of SEQ ID NO:38. The first nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43,SEQ ID NO:47, or SEQ ID NO:51, the second nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:23, the third nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO:33,the fourth nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:9, the fifth nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:21, or SEQ ID NO:49, the sixth nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:27, SEQ ID NO:39, or SEQID NO:53, the seventh nucleotide sequence preferably comprises thenucleotide sequence of SEQ ID NO:25, the eighth nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:13 or SEQ IDNO:31, the ninth nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:7 or SEQ ID NO:11, the tenth nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:41, theeleventh nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO:45, the twelfth nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO:3, and the thirteenthnucleotide sequence preferably comprises the nucleotide sequence of SEQID NO:37. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, and thirteenth polypeptidespreferably are polypeptides involved in post-transcriptional genesilencing.

This invention also includes the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotidepreferably consist of the same number of nucleotides, and the nucleotidesequences of the complement and the polynucleotide preferably have 100%complementarity.

Nucleic acid fragments encoding at least a portion of severalpolypeptides involved in post-transcriptional gene silencing have beenisolated and identified by comparison of random plant cDNA sequences topublic databases containing nucleotide and protein sequences using theBLAST algorithms well known to those skilled in the art. The nucleicacid fragments of the instant invention may be used to isolate cDNAs andgenes encoding homologous proteins from the same or other plant species.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, methods of nucleic acid hybridization, and methodsof DNA and RNA amplification as exemplified by various uses of nucleicacid amplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other polypeptides involved inpost-transcriptional gene silencing, either as cDNAs or genomic DNAs,could be isolated directly by using all or a portion of the instantnucleic acid fragments as DNA hybridization probes to screen librariesfrom any desired plant employing methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the instantnucleic acid sequences can be designed and synthesized by methods knownin the art (Maniatis). Moreover, an entire sequence can be used directlyto synthesize DNA probes by methods known to the skilled artisan such asrandom primer DNA labeling, nick translation, end-labeling techniques,or RNA probes using available in vitro transcription systems. Inaddition, specific primers can be designed and used to amplify a part orall of the instant sequences. The resulting amplification products canbe labeled directly during amplification reactions or labeled afteramplification reactions, and used as probes to isolate full length cDNAor genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 30 (preferably at least 40, most preferably at least 60)contiguous nucleotides derived from a nucleotide sequence of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51 or 53, and the complement of suchnucleotide sequences may be used in such methods to obtain a nucleicacid fragment encoding a substantial portion of an amino acid sequenceof a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another preferred embodiment, this invention includes viruses andhost cells comprising either the recombinant DNA constructs of theinvention as described herein or isolated polynucleotides of theinvention as described herein. Examples of host cells which can be usedto practice the invention include, but are not limited to, yeast,bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of PTGS in thoseplants. Since some of the AGO1 family members have also been shown to beinvolved in transposon silencing, meristem development, anddifferentiation of meristematic tissue, the polynucleotides disclosedherein may also be used to manipulate transposon activity, meristemactivity, plant architecture and development, and proliferation ofundifferentiated plant cells in culture, which would be useful in calluspropagation.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a recombinant DNA construct in whichthe coding region is operably linked to a promoter capable of directingexpression of a gene in the desired tissues at the desired stage ofdevelopment. The recombinant DNA construct may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant recombinant DNA construct may alsocomprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide(s) (orrecombinant DNA construct(s)) may be constructed. The choice of plasmidvector is dependent upon the method that will be used to transform hostplants. The skilled artisan is well aware of the genetic elements thatmust be present on the plasmid vector in order to successfullytransform, select and propagate host cells containing the recombinantDNA construct or chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the recombinant DNAconstruct(s) described above may be further supplemented by directingthe coding sequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), nuclear localization signals (Raikhel(1992) Plant Phys.100:1627-1632) or mitochondrial signal sequences(Zhang and Glaser (2002) Trends Plant Sci 7:14-21) with or withoutremoving targeting sequences that are already present. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of use may be discovered in thefuture.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a recombinant DNA construct designed forco-suppression of the instant polypeptide can be constructed by linkinga gene or gene fragment encoding that polypeptide to plant promotersequences. Alternatively, a recombinant DNA construct designed toexpress antisense RNA for all or part of the instant nucleic acidfragment can be constructed by linking the gene or gene fragment inreverse orientation to plant promoter sequences. Either theco-suppression or antisense recombinant DNA constructs could beintroduced into plants via transformation wherein expression of thecorresponding endogenous genes are reduced or eliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent recombinant DNA constructs utilizing different regulatoryelements known to the skilled artisan. Once transgenic plants areobtained by one of the methods described above, it will be necessary toscreen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

In another preferred embodiment, the present invention includes anisolated polypeptide comprising: (a) a first amino acid sequencecomprising at least 100 amino acids, wherein the first amino acidsequence and the amino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, or SEQID NO:52 have at least 70%, 80%, 85%, 90%, or 95% identity based on theClustaIV alignment method, (b) a second amino acid sequence comprisingat least 200 amino acids, wherein the second amino acid sequence and theamino acid sequence of SEQ ID NO:24 have at least 70%, 80%, 85%, 90%, or95% identity based on the ClustaIV alignment method, (c) a third aminoacid sequence comprising at least 100 amino acids, wherein the thirdamino acid sequence and the amino acid sequence of SEQ ID NO:34 have atleast 80%, 85%, 90%, or 95% identity based on the ClustaIV alignmentmethod, (d) a fourth amino acid sequence comprising at least 150 aminoacids, wherein the fourth amino acid sequence and the amino acidsequence of SEQ ID NO:10 have at least 80%, 85%, 90%, or 95% identitybased on the ClustaIV alignment method, (e) a fifth amino acid sequencecomprising at least 200 amino acids, wherein the fifth amino acidsequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:22, or SEQ ID NO:50 have at least 80%, 85%, 90%, or 95% identitybased on the ClustaIV alignment method, (f) a sixth amino acid sequencecomprising at least 300 amino acids, wherein the sixth amino acidsequence and the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 orSEQ ID NO:54 have at least 80%, 85%, 90%, or 95% identity based on theClustaIV alignment method, (g) a seventh amino acid sequence comprisingat least 100 amino acids, wherein the seventh amino acid sequence andthe amino acid sequence of SEQ ID NO:26 have at least 85%, 90%, or 95%identity based on the ClustaIV alignment method, (h) an eighth aminoacid sequence comprising at least 200 amino acids, wherein the eighthamino acid sequence and the amino acid sequence of SEQ ID NO:14 or SEQID NO:32 have at least 85%, 90%, or 95% identity based on the ClustaIValignment method, (i) a ninth amino acid sequence comprising at least250 amino acids, wherein the ninth amino acid sequence and the aminoacid sequence of SEQ ID NO:8 or SEQ ID NO:12 have at least 85%, 90%, or95% identity based on the ClustaIV alignment method, (j) a tenth aminoacid sequence comprising at least 300 amino acids, wherein the tenthamino acid sequence and the amino acid sequence of SEQ ID NO:42 have atleast 85%, 90%, or 95% identity based on the ClustaIV alignment method,(k) an eleventh amino acid sequence comprising at least 100 amino acids,wherein the eleventh amino acid sequence and the amino acid sequence ofSEQ ID NO:46 have at least 90% or 95% identity based on the ClustaIValignment method, (I) a twelfth amino acid sequence comprising at least150 amino acids, wherein the twelfth amino acid sequence and the aminoacid sequence of SEQ ID NO:4 have at least 90% or 95% identity based onthe ClustaIV alignment method, or (m) a thirteenth amino acid sequencecomprising at least 250 amino acids, wherein the thirteenth amino acidsequence and the amino acid sequence of SEQ ID NO:38 have at least 90%or 95% identity based on the ClustaIV alignment method. The first aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:36, SEQ IDNO:44, SEQ ID NO:48, or SEQ ID NO:52, the second amino acid sequencepreferably comprises the amino acid sequence of SEQ ID NO:24, the thirdamino acid sequence preferably comprises the amino acid sequence of SEQID NO:34, the fourth amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:10, the fifth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:22, or SEQ ID NO:50, the sixth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:28, SEQ ID NO:40 or SEQID NO:54, the seventh amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:26, the eighth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:32, theninth amino acid sequence preferably comprises the amino acid sequenceof SEQ ID NO:8 or SEQ ID NO:12, the tenth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO:42, the eleventh aminoacid sequence preferably comprises the amino acid sequence of SEQ IDNO:46, the twelfth amino acid sequence preferably comprises the aminoacid sequence of SEQ ID NO:4, and the thirteenth amino acid sequencepreferably comprises the amino acid sequence of SEQ ID NO:38. Thepolypeptide preferably is a polypeptide involved in post-transcriptionalgene silencing.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct recombinant DNA constructs for production of the instantpolypeptides. This recombinant DNA construct could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the encoded polypeptides involved in post-transcriptionalgene silencing. An example of a vector for high level expression of theinstant polypeptides in a bacterial host is provided (Example 6).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

Nucleic acid probes derived from the instant nucleic acid sequences maybe used in direct fluorescence in situ hybridization (FISH) mapping(Trask (1991) Trends Genet. 7:149-154). Although current methods of FISHmapping favor use of large clones (several kb to several hundred kb; seeLaan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity mayallow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding one of the instantpolypeptides. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding one of the instant polypeptides can be identified and obtained.This mutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn (Zea mays), soybean(Glycine max), rice (Oryza sativa), and wheat (Triticum aestivum)tissues were prepared. The characteristics of the libraries aredescribed below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean, andWheat Library Tissue Clone cle1f Corn Leaf at VE-V5 Stage**cle1f.pk002.k13 cpf1c Corn Treated with Chemicals cpf1c.pk008.j24Related to Protein Synthesis*** csc1c Corn 20 Day Seedlingcsc1c.pk006.j19 (Germination Cold Stress) ctn1c Corn Tassel, NightHarvested ctn1c.pk003.i20 p0002 Corn Tassel, Premeiotic Cellsp0002.cgevj06r to Early Uninucleate Stage p0102 Corn Early MeiosisTassels* p0102.cerae32ra p0102.cerba57r p0107 Corn Whole Kernels 7 Daysp0107.cbcbd69r After Pollination* p0119 Corn V12 Stage** Ear Shootp0119.cmtmm21r With Husk, Night Harvested* p0125 Corn Anther Prophase I*p0125.czaab55r p0125.czaat57r rca1n Rice Callus* rca1n.pk018.b3 rdi2cRice (Oryza sativa, Nipponbare) rdi2c.pk002.d14 developing inflorescenceat rachis branch-floral organ primordia formation rl0n Rice 15 Day OldLeaf* rl0n.pk124.g8 rlm1n Rice Leaf 15 Days After rlm1n.pk001.m11Germination, Harvested 2-72 Hours Following Infection With Magnaportagrisea (4360-R-62 and 4360-R-67)* rls6 Susceptible Rice Leaf 15rls6.pk0082.d4 Days After Germination, 6 Hours After Infection of StrainMagnaporthe grisea 4360-R-67 (AVR2-YAMO) rsl1n Rice 15-Day-Old Seedling*rsl1n.pk004.d12 rtc1c Rice Leaf Inoculated with rtc1c.pk008.k19.fMagnaporthe grisea Strain 0184 at 4, 8, and 24 Hours sdc1c SoybeanDeveloping Cotyledon (3-5 mm) sdc1c.pk0004.d11 sdc2c Soybean DevelopingCotyledon (6-7 mm) sdc2c.pk001.p4 ses2w Soybean Embryogenicses2w.pk0009.g6 Suspension 2 Weeks After Subculture ssl1c Soybean Seed25 Days After ssl1c.pk003.g3 Fertilization ssm Soybean Shoot Meristemssm.pk0063.a4 wdk1c Wheat Developing Kernel, wdk1c.pk012.i2 3 Days AfterAnthesis wlm96 Wheat Seedlings 96 Hours wlm96.pk029.c23 AfterInoculation With Erysiphe graminis f. sp tritici wne1g Wheat NebulizedGenomic Library wne1g.pk003.f8 wr1 Wheat Root From 7 Day Oldwr1.pk0073.c7 Light Grown Seedling wre1n Wheat Root From 7 Day Oldwre1n.pk0001.h6 Etiolated Seedling* wre1n.pk162.h10*These libraries were normalized essentially as described in U.S. Pat.No. 5,482,845, incorporated herein by reference.**Corn developmental stages are explained in the publication “How a cornplant develops” from the Iowa State University Coop. Ext. ServiceSpecial Report No. 48 reprinted June 1993.***Chemicals used included chloramphenicol, cyclohexamide,aurintricarboxylic acid, all of which are commercially available fromCalbiochem-Novabiochem Corp. (1-800-628-8470)

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phred/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

In some of the clones the cDNA fragment corresponds to a portion of the3′-terminus of the gene and does not cover the entire open readingframe. In order to obtain the upstream information one of two differentprotocols are used. The first of these methods results in the productionof a fragment of DNA containing a portion of the desired gene sequencewhile the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

cDNA clones encoding polypeptides involved in post-transcriptional genesilencing were identified by conducting BLAST (Basic Local AlignmentSearch Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsothe explanation of the BLAST algorithm on the world wide web site forthe National Center for Biotechnology Information at the NationalLibrary of Medicine of the National Institutes of Health) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The cDNA sequences obtained in Example 1 were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Polypeptides Involvedin Post-Transcriptional Gene Silencing

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs topolypeptides involved in post-transcriptional gene silencing and AGO1family members from Neurospora crassa (NCBI GenBank Identifier (GI) No.7248733), Arabidopsis thaliana (NCBI GI Nos. 3885334, 6692120, 11386626,2149640, 5107374, 12643935 and 15221177), and Oryza sativa (NCBI GI No.6539559). The following three Arabidopsis thaliana sequences eachrepresent the same 1048 amino acid sequence: GI No. 11386626; GI No.2149640; and GI No. 15221177. The following two Arabidopsis thalianasequences each represent the same 988 amino acid sequence: GI No.5107374 and GI No. 12643935. Shown in Table 3 are the BLAST results forindividual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), the sequences of contigsassembled from two or more EST, FIS or PCR sequencess (“Contig”), orsequences encoding an entire protein, or functionally activepolypeptide, derived from an FIS or a contig (“CGS”): TABLE 3 BLASTResults for Sequences Encoding Polypeptides Homologous to PolypeptidesInvolved in Post-Transcriptional Gene Silencing (AGO1 Protein Family)BLAST Results BLAST NCBI pLog Clone Status GI No. Score p0102.cerba57rFIS 12643935 >180.00 ses2w.pk0009.g6 FIS 5107374 >180.00 ssm.pk0063.a4FIS 5107374 >180.00 cle1f.pk002.k13 (FIS) CGS 6539559 >180.00cpf1c.pk008.j24 FIS 2149640 >180.00 csc1c.pk006.j19 (FIS) CGS2149640 >180.00 ctn1c.pk003.i20 (FIS) CGS 2149640 >180.00 Contig ofContig 11386626 >180.00 p0002.cgevj06r p0125.czaab55r (FIS)p0125.czaat57r p0102.cerae32ra EST 5107374 31.10 p0107.cbcbd69r EST2149640 57.15 p0119.cmtmm21r (FIS) CGS 6539559 >180.00 rca1n.pk018.b3FIS 2149640 >180.00 rl0n.pk124.g8 FIS 2149640 131.00 Contig of CGS11386626 >180.00 rlm1n.pk001.m11 (FIS) PCR fragment sequencerls6.pk0082.d4 FIS 6539559 31.70 rsl1n.pk004.d12 FIS 11386626 171.00rtc1c.pk008.k19.f EST 2149640 64.22 sdc1c.pk0004.d11 FIS 6692120 76.05Contig of CGS 2149640 >180.00 sdc2c.pk001.p4 (FIS) PCR fragment sequencessl1c.pk003.g3 (FIS) CGS 3885334 >180.00 Contig of CGS 6539559 >180.00wdk1c.pk012.i2 (FIS) PCR fragment sequence wlm96.pk029.c23 FIS 724873345.30 wne1g.pk003.f8 EST 2149640 47.10 wr1.pk0073.c7 EST 2149640 27.70wre1n.pk0001.h6 FIS 6539559 >180.00 wre1n.pk162.h10 EST 2149640 30.70rdi2c.pk002.d14 (FIS) CGS 15221177 >180.00

FIGS. 1A-1D present an alignment of the amino acid sequences set forthin SEQ ID NOs:8, 22, 40, and 42, and the Oryza sativa sequence (NCBI GINo. 6539559; SEQ ID NO:55). FIGS. 2A-2E present an alignment of theamino acid sequences set forth in SEQ ID NOs:12, 14, 28, and 38, and theArabidopsis thaliana sequence (NCBI GI No. 2149640; SEQ ID NO:56). Thedata in Table 5 represents a calculation of the percent identity of theamino acid sequences set forth in SEQ ID NOs:8, 12, 14, 22, 28, 38, 40,and 42, the Oryza sativa sequence (NCBI GI No. 6539559; SEQ ID NO: 55),and the Arabidopsis thaliana sequence (NCBI GI No. 2149640; SEQ ID NO:56). TABLE 5 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences Encoding Polypeptides Homologous to PolypeptidesInvolved in Post-Transcriptional Gene Silencing (AGO1 Protein Family)SEQ ID NO. NCBI GI No. Percent Identity 8 6539559 82.2 12 2149640 72.114 2149640 72.6 22 6539559 73.2 28 2149640 72.2 38 2149640 78.2 406539559 68.8 42 6539559 83.7 54 2149640 73.3

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the ClustaIV method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe ClustaIV method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a polypeptide involved inpost-transcriptional gene silencing. These sequences represent the firstcorn and wheat sequences indicated to encode polypeptides involved inpost-transcriptional gene silencing (AGO1 protein family) known toApplicants.

Example 4 Expression of Recombinant DNA Constructs in Monocot Cells

A recombinant DNA construct comprising a cDNA encoding the instantpolypeptide in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone, plant cDNA or plant cDNA libraries usingappropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can beincorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the digested vector pML103 asdescribed below. Amplification is then performed in a standard PCR. Theamplified DNA is then digested with restriction enzymes NcoI and SmaIand fractionated on an agarose gel. The appropriate band can be isolatedfrom the gel and combined with a 4.9 kb NcoI-SmaI fragment of theplasmid pML103. Plasmid pML103 has been deposited under the terms of theBudapest Treaty at ATCC (American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209), and bears accession numberATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoIpromoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalIfragment from the 3′ end of the maize 10 kD zein gene in the vectorpGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C.overnight, essentially as described (Maniatis). The ligated DNA may thenbe used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™;Stratagene). Bacterial transformants can be screened by restrictionenzyme digestion of plasmid DNA and limited nucleotide sequence analysisusing the dideoxy chain termination method (Sequenase™ DNA SequencingKit; U.S. Biochemical). The resulting plasmid construct would comprise arecombinant DNA construct encoding, in the 5′ to 3′ direction, the maize27 kD zein promoter, a cDNA fragment encoding the instant polypeptide,and the 10 kD zein 3′ region.

The recombinant DNA construct described above can then be introducedinto corn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the³⁵S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialaphos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialaphos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialaphos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Plants in which PTGS has been elevated or diminished can be assayed bymaking the following two sexual crosses: (1) a first transgenic plant,transformed with a gene encoding a polypeptide involved in PTGS, iscrossed with a second transgenic plant that contains an active reportertransgene, such as the GUS gene, and (2), the first transgenic plant iscrossed with a third transgenic plant that contains apost-transcriptionally silenced reporter gene. If PTGS has beenelevated, reporter gene expression in the progeny plants from the firstcross should be reduced. If PTGS has been diminished, reporter geneexpression in progeny plants from the second cross should be increased.Also, if PTGS has been diminished, a correlated decrease in themethylation state of the reporter transgene in the progeny of the secondcross would be expected (Fagard et al. (2000) Proc Natl Acad Sci USA97:11650-11654).

Example 5 Expression of Recombinant DNA Constructs in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites NcoI (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by HindIII sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries,using appropriate oligonucleotide primers. Cloning sites can beincorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the expression vector. Amplificationis then performed as described above, and the isolated fragment isinserted into a pUC18 vector carrying the seed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the ³⁵S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Recombinant DNA Constructs in Microbial Cells

The cDNA fragment of the gene may be generated by polymerase chainreaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries,using appropriate oligonucleotide primers. The cDNAs encoding theinstant polypeptides can be inserted into the T7 E. coli expressionvector pBT430. This vector is a derivative of pET-3a (Rosenberg et al.(1987) Gene 56:125-135) which employs the bacteriophage T7 RNApolymerase/T7 promoter system. Plasmid pBT430 was constructed by firstdestroying the EcoRI and HindIII sites in pET-3a at their originalpositions. An oligonucleotide adaptor containing EcoRI and Hind IIIsites was inserted at the BamHI site of pET-3a. This created pET-3aMwith additional unique cloning sites for insertion of genes into theexpression vector. Then, the NdeI site at the position of translationinitiation was converted to an NcoI site using oligonucleotide-directedmutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, wasconverted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 7 Expression of Recombinant DNA Constructs in Yeast Cells

The polypeptides encoded by the polynucleotides of the instant inventionmay be expressed in a yeast (Saccharomyces cerevisiae) strain YPH.Plasmid DNA, plant cDNA or plant cDNA libraries, may be used as templateto amplify the portion encoding the polypeptide involved inpost-transcriptional gene silencing. Amplification may be performedusing the GC melt kit (Clontech) with a 1 M final concentration of GCmelt reagent and using a Perkin Elmer 9700 thermocycler. The amplifiedinsert may then be incubated with a modified pRS315 plasmid (NCBIGeneral Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989)Genetics 122:19-27) that has been digested with Not I and Spe I. PlasmidpRS315 has been previously modified by the insertion of a bidirectionalgal1/10 promoter between the Xho I and Hind III sites. The plasmid maythen be transformed into the YPH yeast strain using standard procedureswhere the insert recombines through gap repair to form the desiredtransformed yeast strain (Hua, S. B. et al. (1997) Plasmid 38:91-96).

Yeast cells may be prepared according to a modification of the methodsof Pompon et al. (Pompon, D. et al. (1996) Meth. Enz. 272:51-64).Briefly, a yeast colony will be grown overnight (to saturation) in SG(-Leucine) medium at 30° C. with good aeration. A 1:50 dilution of thisculture will be made into 500 mL of YPGE medium with adeninesupplementation and allowed to grow at 30° C. with good aeration to anOD₆₀₀ of 1.6 (24-30 h). Fifty mL of 20% galactose will be added, and theculture allowed to grow overnight at 30° C. The cells will be recoveredby centrifugation at 5,500 rpm for five minutes in a Sorvall GS-3 rotor.The cell pellet resuspended in 500 mL of 0.1 M potassium phosphatebuffer (pH 7.0) and then allowed to grow at 30° C. for another 24 hours.

The cells may be recovered by centrifugation as described above and thepresence of the polypeptide of the instant invention determined byHPLC/mass spectrometry or any other suitable method.

Example 8 Expression of Recombinant DNA Constructs in Insect Cells

The cDNA fragment of the gene may be generated by polymerase chainreaction (PCR) of the cDNA clone, plant cDNA or plant cDNA libraries,using appropriate oligonucleotide primers. The cDNAs encoding theinstant polypeptides may be introduced into the baculovirus genomeitself. For this purpose the cDNAs may be placed under the control ofthe polyhedron promoter, the IE1 promoter, or any other one of thebaculovirus promoters. The cDNA, together with appropriate leadersequences is then inserted into a baculovirus transfer vector usingstandard molecular cloning techniques. Following transformation of E.coli DH5α, isolated colonies are chosen and plasmid DNA is prepared andis analyzed by restriction enzyme analysis. Colonies containing theappropriate fragment are isolated, propagated, and plasmid DNA isprepared for cotransfection.

Spodoptera frugiperda cells (Sf-9) are propagated in ExCell® 401 media(JRH Biosciences, Lenexa, Kans.) supplemented with 3.0% fetal bovineserum. Lipofectin® (50 μL at 0.1 mg/mL, Gibco/BRL) is added to a 50 μLaliquot of the transfer vector containing the toxin gene (500 ng) andlinearized polyhedrin-negative AcNPV (2.5 μg, Baculogold® viral DNA,Pharmigen, San Diego, Calif.). Sf-9 cells (approximate 50% monolayer)are co-transfected with the viral DNA/transfer vector solution. Thesupernatant fluid from the co-transfection experiment is collected at 5days post-transfection and recombinant viruses are isolated employingstandard plaque purification protocols, wherein only polyhedrin-positiveplaques are selected (O'Reilly et al. (1992), Baculovirus ExpressionVectors: A Laboratory Manual, W. H. Freeman and Company, New York.).Sf-9 cells in 35 mM petri dishes (50% monolayer) are inoculated with 100μL of a serial dilution of the viral suspension, and supernatant fluidsare collected at 5 days post infection. In order to prepare largerquantities of virus for characterization, these supernatant fluids areused to inoculate larger tissue cultures for large-scale propagation ofrecombinant viruses.

1. An isolated polynucleotide comprising: (a) a first nucleotidesequence encoding a first polypeptide having post-transcriptional genesilencing activity, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO:12, 14, 22, 28, 40or 54 have at least 80% sequence identity based on the ClustaIValignment method, (b) a second nucleotide sequence encoding a secondpolypeptide having post-transcriptional gene silencing activity, whereinthe amino acid sequence of the second polypeptide and the amino acidsequence of SEQ ID NO:8, 38 or 42 have at least 85% sequence identitybased on the ClustaIV alignment method, or (c) the complement of thenucleotide sequence of (a) or (b).
 2. The polynucleotide of claim 1,wherein the amino acid sequence of the first polypeptide and the aminoacid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 85%sequence identity based on the ClustaIV alignment method.
 3. Thepolynucleotide of claim 1, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO: 12, 14, 22, 28, 40or 54 have at least 90% sequence identity based on the ClustaIValignment method, and wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have atleast 90% sequence identity based on the ClustaIV alignment method. 4.The polynucleotide of claim 1, wherein the amino acid sequence of thefirst polypeptide and the amino acid sequence of SEQ ID NO: 12, 14, 22,28, 40 or 54 have at least 95% sequence identity based on the ClustaIValignment method, and wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO:8, 38 or 42 have atleast 95% sequence identity based on the ClustaIV alignment method. 5.The polynucleotide of claim 1, wherein the amino acid sequence of thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:12, 14,22, 28, 40 or 54, and wherein the amino acid sequence of the secondpolypeptide comprises the amino acid sequence of SEQ ID NO:8, 38 or 42.6. The polynucleotide of claim 1, wherein the first nucleotide sequencecomprises the nucleotide sequence of SEQ ID NO:11, 13, 21, 27, 39 or 53,and wherein the second nucleotide sequence comprises the nucleotidesequence of SEQ ID NO:7, 37 or
 41. 7. A vector comprising thepolynucleotide of claim
 1. 8. A recombinant DNA construct comprising thepolynucleotide of claim 1 operably linked to at least one regulatorysequence.
 9. A method for transforming a cell, comprising transforming acell with the polynucleotide of claim
 1. 10. A cell comprising therecombinant DNA construct of claim
 8. 11. A method for production of apolypeptide having post-transcriptional gene silencing activitycomprising the steps of cultivating the cell of claim 10 underconditions that allow for the synthesis of the polypeptide and isolatingthe polypeptide from the cultivated cells, from the culture medium, orfrom both the cultivated cells and the culture medium.
 12. A method forproducing a plant comprising transforming a plant cell with thepolynucleotide of claim 1 and regenerating a plant from the transformedplant cell.
 13. A plant comprising the recombinant DNA construct ofclaim
 8. 14. A seed comprising the recombinant DNA construct of claim 8.15. An isolated polypeptide having post-transcriptional gene silencingactivity, wherein the polypeptide comprises: (a) a first amino acidsequence, wherein the first amino acid sequence and and the amino acidsequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 80%sequence identity based on the ClustaIV alignment method, or (b) asecond amino acid sequence, wherein the second amino acid sequence andand the amino acid sequence of SEQ ID NO:8, 38 or 42 have at least 85%sequence identity based on the ClustaIV alignment method.
 16. Thepolypeptide of claim 15, wherein the first amino acid sequence and theamino acid sequence of SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least85% sequence identity based on the ClustaIV alignment method.
 17. Thepolypeptide of claim 15, wherein the first amino acid sequence and theamino acid sequence SEQ ID NO:12, 14, 22, 28, 40 or 54 have at least 90%sequence identity based on the ClustaIV alignment method, and whereinthe second amino acid sequence and the amino acid sequence of SEQ IDNO:8, 38 or 42 have at least 90% sequence identity based on the ClustaIValignment method.
 18. The polypeptide of claim 15, wherein the firstamino acid sequence and the amino acid sequence SEQ ID NO:12, 14, 22,28, 40 or 54 have at least 95% sequence identity based on the ClustaIValignment method, and wherein the second amino acid sequence and theamino acid sequence of SEQ ID NO:8, 38 or 42 have at least 95% sequenceidentity based on the ClustaIV alignment method.
 19. The polypeptide ofclaim 15, wherein the first amino acid sequence comprises the amino acidsequence of SEQ ID NO:12, 14, 22, 28, 40 or 54, and wherein the secondamino acid sequence comprises the amino acid sequence of SEQ ID NO:8, 38or 42.